GSM7926092: Germinal Center B cells - MycGFP positive ATAC; Mus musculus; ATAC-seq
Splenic B cells from mice immunized with SRBC for 9 days were pre-enriched for B220pos cells by negative magnetic enrichment and stained for GC markers as described above before flow sorting. GC Myc-GFPpos and Myc-GFPneg fractions were sorted from 6 individual 3-month-old male mice and pooled before processing for Multiomic sequencing using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10X Genomics #N-1000283) according to the provider specifications. Briefly, two independent nuclei suspensions (one per Myc-GFP fraction) were prepared following the Low Cell Input protocol with confirmed Countess II Live/Dead ratio of <2% with a 3min lysis. Transposition was performed for 1h at 37°C, after which samples were loaded onto the 10x Genomics Chromium platform to generate barcoded single-cell GEMs, targeting about 10000 nuclei recovered (about 8000 nuclei/μl) per sample. GEM incubation (37 °C for 45 min, 25 °C for 30 min; held at 4 °C) was performed in a C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module (Bio- Rad) for the 10X barcode attachment as well as reverse transcription, template switch and extension. After the RT reaction, GEMs were broken up and the single strand cDNAs were cleaned up with DynaBeads MyOne Silane Beads (ThermoFisher; 37002D). Pre-Amplification (72 °C for 5 min; 98 °C for 3 min, 98 °C for 20 s, 63 °C for 30 s, 72 °C for 1 min for 7 cycles followed by 72 °C for 1 min; held at 4 °C) was performed before proceeding to the ATAC (A) and Gene expression (B) library constructions. (A) A unique sample index for each library was introduced to the pre-amplified tagmented DNA through 7 cycles of PCR amplification using the indexes provided in the kit (98 °C for 45 s; 98 °C for 20 s, 67 °C for 30 s, and 72 °C for 20 s x 7 cycles; 72 °C for 1 min; held at 4 °C). Indexed libraries were subjected to a second double-sided size selection, and libraries were then quantified using Qubit (ThermoFisher, RRID:SCR_020553). The quality was assessed on an Agilent Bioanalyzer 2100 (RRID:SCR_019389), obtaining an average library size of 441 (GC Myc-GFPneg) and 405 (Myc-GFPpos) bp. (B) The GC Myc-GFPneg and Myc-GFPpos cDNAs were amplified for 6 cycles (98 °C for 3 min; 98 °C for 15 s, 63 °C for 20 s, 72 °C for 1 min, 72 °C for 1 min; held at 4 °C). Quality of the cDNAs was assessed using Agilent Bioanalyzer 2100, obtaining an average product size of 1472 and 1503 bp, respectively. These cDNAs were enzymatically fragmented, end repaired, A-tailed, subjected to a double-sided size selection with SPRIselect beads (Beckman Coulter) and ligated to adaptors provided in the kit. A unique sample index for each library was introduced through 15 cycles of PCR amplification using the indexes provided in the kit (98 °C for 45 s; 98 °C for 20 s, 54 °C for 30 s, and 72 °C for 20 s x 14 cycles; 72 °C for 1 min; held at 4 °C). Indexed libraries were subjected to a second double-sided size selection, and libraries were then quantified using Qubit (ThermoFisher, RRID:SCR_020553). The quality was assessed on an Agilent Bioanalyzer 2100 (RRID:SCR_019389), obtaining an average library size of 441 (GC Myc-GFPneg) and 455 bp (GC Myc-GFPpos). Libraries were diluted to 2 nM and clustered on an Illumina NovaSeq 2000 (RRID:SCR_016387) on a pair-end read flow cell and sequenced for: (A) 50 cycles on R1 (10x barcode and the UMIs), followed by 8 cycles of i7 Index and 24 cycles of i5 Index (sample Index) and 49 bases on R2, with a coverage around 200M reads per sample. (B)28 cycles on R1 (10x barcode and the UMIs), followed by 10 cycles of i7 Index and 10 cycles of i5 Index (sample Index) and 90 bases on R2, with a coverage around 200M reads per sample.  Primary processing of sequencing images was done using Illumina's Real Time Analysis software (RTA). Other: 10X Multiome kit: https://www.10xgenomics.com/products/single-cell-multiome-atac-plus-gene-expression